U.S. patent number 5,658,795 [Application Number 08/571,739] was granted by the patent office on 1997-08-19 for method for biodegradation of polluting substance.
This patent grant is currently assigned to Canon Kabushiki Kaisha. Invention is credited to Kinya Kato, Shinya Kozaki, Masanori Sakuranaga, Kazumi Tanaka.
United States Patent |
5,658,795 |
Kato , et al. |
August 19, 1997 |
**Please see images for:
( Certificate of Correction ) ** |
Method for biodegradation of polluting substance
Abstract
A method of biodegradation of a polluting substance by a
microorganism is disclosed, wherein the microorganism is an
auxotrophic microorganism and the action of the auxotrophic
microorganism is controlled by the amount of a required nutrient
for the auxotrophic microorganism. A carrier for supporting an
auxotrophic microorganism for use for biodegradation contains a
required nutrient for the auxotrophic microorganism.
Inventors: |
Kato; Kinya (Yokohama,
JP), Tanaka; Kazumi (Yokohama, JP),
Sakuranaga; Masanori (Atsugi, JP), Kozaki; Shinya
(Kawasaki, JP) |
Assignee: |
Canon Kabushiki Kaisha (Tokyo,
JP)
|
Family
ID: |
17646662 |
Appl.
No.: |
08/571,739 |
Filed: |
December 13, 1995 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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142068 |
Oct 20, 1993 |
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Foreign Application Priority Data
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Oct 20, 1992 [JP] |
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4-281987 |
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Current U.S.
Class: |
435/262.5;
435/267; 435/262 |
Current CPC
Class: |
C02F
3/108 (20130101); B09C 1/10 (20130101); C02F
3/34 (20130101); Y02W 10/15 (20150501); Y02W
10/10 (20150501) |
Current International
Class: |
B09C
1/10 (20060101); C02F 3/34 (20060101); A62D
3/00 (20060101); C12N 001/20 (); C07C 061/00 () |
Field of
Search: |
;435/262,262.5,267 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Finette et al., "Isolation and Characterization of Pseudomonas
Putida . . . ", 1984, J. Bacter., vol. 160, p. 1003-9. .
Wackett et al., "Degradation of Trichloroethylene by Toluene . . .
", 1988, App. Env. Microbial., vol. 54, pp. 1703-1708. .
Crueger et al. Biotechnology: Textbook of Industrial Microbiology
pp, 131-139, Science Tech, Madison, 1982. .
APS Japanese Patent Office Abstract 03-251178 Svemitsu, R. Nov. 8,
1991..
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Primary Examiner: Paden; Carolyn
Attorney, Agent or Firm: Fitzpatrick, Cella, Harper &
Scinto
Parent Case Text
This application is a continuation of application Ser. No.
08/142,068 filed Oct. 20, 1993, now abandoned.
Claims
What is claimed is:
1. A method of biodegradation of a polluting substance in an open
ecosystem, comprising the steps of:
(a) providing a strain selected from the group consisting of
auxotrophic mutants of Pseudomonas cepacia KK01, deposited as
FERM-4235, possessing the capability of the parent organism for
degrading phenol and cresol;
(b) applying the auxotrophic strain into the ecosystem in the
presence of an organic nutrient required by the auxotrophic strain;
and
(c) controlling the growth of the auxotrophic strain in the
ecosystem by providing sufficient nutrient to the auxotrophic
strain to effect the biodegradation of the polluting substance.
2. The method for biodegradation according to claim 1, including
conducting the biodegradation of the polluting substance in an
aqueous environment.
3. The method for biodegradation according to claim 1, including
conducting the biodegradation of the polluting substance in a soil
environment.
4. The method of biodegradation according to any of claims 2, 3, or
7 wherein the Pseudomonas cepacia is supported on a carrier.
5. A method of biodegradation of a polluting substance in an open
ecosystem, comprising the steps of:
(a) providing a strain selected from the group consisting of
auxotrophic mutants of Pseudomonas cepacia KK01, deposited as
FERM-4235, possessing the capability of the parent organism for
degrading phenol and cresol;
(b) applying the auxotrophic strain into the ecosystem;
(c) periodically adding sufficient amounts of an organic nutrient
required by the auxotrophic strain to stimulate growth of the
auxotrophic strain and effect biodegradation of the polluting
substance; and
(d) terminating said addition of nutrient to inhibit the growth of
the auxotrophic strain.
6. The method for biodegradation according to claim 5, including
conducting the biodegradation of the polluting substance in an
aqueous environment.
7. The method for biodegradation according to claim 5, including
conducting the biodegradation of the polluting substance in a soil
environment.
8. The method of degradation according to any of claims 5-7
including supporting the auxotrophic strain on a carrier.
9. A carrier for remediating a polluted open ecosystem which
comprises;
(a) a synthetic support;
(b) a strain selected from the group consisting of auxotrophic
mutants of pseudomonas cepacia KK01, deposited as FERM-4235,
possessing the capability of the parent organism for degrading
phenol and cresol, which is attached to said support; and
(c) a nutrient required for stimulating growth of said auxotrophic
strain, said nutrient contained in said support.
10. A method of biodegrading a pollutant comprising a phenolic
compound in an open ecosystem comprising the steps of:
(a) selecting an auxotrophic mutant of a pollutant degradable
microorganism capable of degrading the pollutant;
(b) applying the auxotrophic mutant into the ecosystem;
(c) providing an organic nutrient which is essential for the
auxotrophic mutant to grow, to the auxotrophic mutant in the
ecosystem to carry out the biodegradation of the pollutant; and
(d) terminating supply of said nutrient to cause the auxotrophic
mutant in the ecosystem to become exhausted after completing the
biodegradation of the pollutant.
11. The method according to claim 10, wherein the nutrient is amino
acid.
12. The method according to claim 11, wherein the amino acid is
leucine.
13. A method of biodegrading a pollutant in an open ecosystem
comprising the steps of:
(a) providing an auxotrophic mutant strain of Pseudomonas cepacia,
the auxotrophic mutant strain being capable of degrading the
pollutant;
(b) applying the auxotrophic mutant strain into the ecosystem;
(c) providing an organic nutrient which is essential for the
auxotrophic mutant strain to grow, to the auxotrophic mutant strain
in the ecosystem to carry out the biodegradation of the pollutant;
and
(d) terminating supply of said nutrient to cause the auxotrophic
mutant strain in the ecosystem to become exhausted after completing
the biodegradation of the pollutant.
14. The method according to claim 13, wherein the organic nutrient
is amino acid.
15. A method according to claim 13, wherein the amino acid is
leucine.
16. A method for remedying an area polluted with a pollutant
comprising a phenolic compound in an environment comprising the
steps of:
(a) selecting an auxotrophic mutant of a pollutant degradable
microorganism capable of degrading the pollutant;
(b) applying the auxotrophic mutant into the environment;
(c) supplying a nutrient which is essential for the auxotrophic
mutant to grow, only into the polluted area to carry out the
biodegradation of the pollutant in the area; and
(d) terminating supply of said nutrient to cause the auxotrophic
mutant in the area to become exhausted after completing the
biodegradation of the pollutant in the area.
17. A method according to claim 16, wherein the nutrient is
supplied by adding a carrier containing the nutrient.
18. A method according to claim 17, wherein the carrier supports
the auxotrophic mutant.
19. A method according to claim 16, wherein the biodegradation of
the pollutant is conducted in a liquid.
20. A method according to claim 16, wherein the biodegradation of
the pollutant is conducted in a soil.
21. A method according to any one of claims 16-20, wherein the
auxotrophic mutant is an auxotrophic Pseudomonas cepacia.
22. A method according to claim 21, wherein the auxotrophic mutant
is an auxotrophic strain of Pseudomonas cepacia KK01, deposited as
FERM BP-4235.
23. A method according to claim 16, wherein the nutrient is amino
acid.
24. A method according to claim 23, wherein the amino acid is
leucine.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method of biodegradation of a
polluting substance, which method employs an auxotrophic
microorganism and is useful for environment protection.
2. Related Background Art
In recent years, various harmful and non-decomposable chemical
substances have come to be detected in soil, river, sea, air and
other environments, and the environmental pollution has become
highlighted. The adverse effects given by such substances are
become a matter of great concern. Therefore, prevention of
pollution and remediation of the environment from pollution are
strongly demanded. An example of environment remediation technique
is a strengthening of functions of microorganisms in the ecosystem
to decompose the polluting substances into non-polluting
substances. This technique intends to stimulate the natural
degradation processes, to promote the decomposition of the
polluting substance. This technique is further advanced to
introduce intentionally from the outside a microorganism having an
ability of decompose a polluting substance to promote remediation
of a polluted area to a non-polluted state. Biodegradative clean-up
technology is demanded in various fields such as treatment of
polluted soil in gas production plant sites, oil refinery plant
sites, sites of a demolished petroleum refinery, sites of a
demolished fuel stockyard, sites of a demolished pulp plant;
cleaning of lakes and marshes, rivers, sea water, underground
water, and the like; treatment of drinking water and industrial
water; treatment of industrial waste water and living waste water;
and so forth. Pollution of soil may cause spread of the polluted
area by diffusion of the polluting substance with underground
water, and pollution of river or the like may cause spread of the
polluting substance with flow of water over the basin or the shore.
Therefore, an effective method for removal of polluting substance
from soil and water is strongly demanded.
Although various physical and chemical methods are known for
removal of polluting substances, few of the methods are
satisfactory from a standpoint of cost, ease of operation, and
decomposition efficiency. For soil treatment, for example, a vacuum
extraction method is available which sucks out the polluting
substance from the soil. This method; however, is unsatisfactory in
view of the cost, ease of the operation, and the efficiency of
elimination of the polluting substance.
Therefore, a biodegradation method is attracting attention to
offset the disadvantages of the physical and chemical methods.
However, if a microorganism for biodegradation is applied in a high
concentration to soil or river, the ecosystem in the applied area
will be changed. Such change of ecosystem is not favorable for
environmental protection. The ecosystem in the treated area should
desirably be restored to the original state when the polluting
substance has been removed or decomposed. Furthermore, if the
microorganism diffuses out unnecessarily from the polluted area
during the biodegradation, the diffused microorganism itself may
possibly cause secondary pollution or other hazard. Ideally, the
microorganism introduced from the outside should work only in the
polluted area, and become extinct spontaneously after the
environment is remediated. In particular, in use of a microorganism
in open systems such as soil, lake, marsh, river and sea, the fate
of the microorganism is a great problem.
SUMMARY OF THE INVENTION
The present invention intends to provide a method of biodegradation
of a polluting substance, where an applied microorganism works only
in a polluted area and becomes extinct spontaneously after the
remediation of the pollution so as to avert the effect of the
remaining microorganism on the ecosystem.
The present invention provides a method of biodegradation of a
polluting substance by a microorganism, the microorganism being an
auxotrophic microorganism and the action of the auxotrophic
microorganism being controlled by the amount of a required nutrient
for the auxotrophic microorganism.
The present invention also provides a carrier for supporting an
auxotrophic microorganism for use for biodegradation, the carrier
containing a required nutrient for the auxotrophic
microorganism.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph showing the results obtained in Example 1.
FIG. 2 is a graph showing the results obtained in Example 2 and
Comparative Example 1.
FIG. 3 is a graph showing the results obtained in Example 3 and
Comparative Example 2.
FIG. 4 is a graph showing the results obtained in Example 4 and
Comparative Example 3.
FIG. 5 is a graph showing the results obtained in Example 5 and
Comparative Example 4.
FIG. 6 is a graph showing the results obtained in Example 6.
FIG. 7 is a graph showing the results obtained in Comparative
Example 5.
FIG. 8 illustrates a state of the layer of the soil in Example
7.
FIG. 9 is a graph showing the results obtained in Example 7.
FIG. 10 is a graph showing the results obtained in Example 7.
FIG. 11 is a graph showing the results obtained in Example 8 and
Comparative Example 6.
DESCRIPTION OF THE PREFERRED EMBODIMENT
The inventors of the present invention noticed the application of
an auxotrophic microorganism to remediation of an ecosystem as
described above. The inventors found that when found the applied
auxotrophic microorganism is allowed to work only in the polluted
area or the area where the microorganism is made to work, then the
microorganism becomes extinct spontaneously after the remediation
of the pollution. This result is obtained when the action of the
auxotrophic microorganism is controlled by adjusting an amount of
the substance required by the auxotrophic microorganism
(hereinafter referred to as a "required nutrient"). Thus the
present invention has been completed.
A microorganism, which has lost a metabolic system or biosynthetic
system of a certain nutrient, is incapable of producing the
nutrient in the cell of the microorganism, and requires a supply
from the outside of the nutrient necessary for living and
multiplying. Examples of such required nutrients include amino
acids, nucleic acid bases, vitamins, organic acids, and other
growth factors. Without the supply of the required nutrient, the
microorganism will become extinct. If such an auxotrophic
microorganism is employed for environment remediation, the
microorganism will survive in the area during the time when the
activity of the microorganism is needed as long as the required
nutrient coexists. The diffusion or growth of the microorganism
will be stopped either by limiting the supply of the required
nutrient to a specific area or by limiting the time the nutrient is
supplied.
The method of the present invention is applicable generally to any
kind of microorganism. The kind of the microorganism to be employed
may be suitably selected in accordance with the properties of the
chemical substance to be decomposed and removed. For example,
Pseudomonas sp., Acinetobacter sp., Metyiosinus sp., and the like
which exhibit activities of pollutant-decomposition are suitable
for removal of dyes having an aromatic ring or furan structure,
pigments, surfactants, surface-coating agents, adhesives, organic
solvents, petroleum type pollutants, etc.
The kind of the auxotrophic microorganism is not particularly
limited, and those which require a nutrient such as an amino acid
(e.g., leucine, tryptophane, histidine, arginine, etc.), a nucleic
acid base(e.g. thymine), a vitamin, an organic acid, or the like
are useful. It is desirable that the kind of the auxotrophic
microorganism is selected so as to meet suitably the conditions of
the environment. Such an auxotrophic microorganism can be derived
by subjecting a microorganism to ultraviolet light irradiation,
chemical treatment with nitrosoguanidine, or the like treatment in
a conventional manner. For example, methods of the treatment are
described in the book: "Biseibutsu Jikkengaku (Experiment in
microbiology)" pp. 288-306, (Kodansha Scientific K.K.). The
nutrient may be in any state of a liquid, a solid, a fluid, or the
like provided that it is readily usable to the auxotrophic
microorganism.
The auxotrophic microorganism is usually applied to the polluted
environment in a form of a bacterial mass in a conventional manner.
The treatment with the applied microorganism may be conducted in
the co-existence of a carrier for the microorganism. Herein the
co-existence includes the cases: (a) the carrier is supplied to the
polluted environment separately before or after the application of
the microorganism, and (b) the microorganism to be applied is
preliminarily fixed on the carrier.
In the case of (b), the microorganism may be fixed by a
conventional immobilization method: including solid bonding
(physical adsorption, ionic adsorption, covalent bonding, etc.) in
which the microorganism is directly or indirectly bonded to the
surface of an organic or inorganic water-insoluble carrier such as
cellulose, nylon, and ceramic; bridging by a compound having two or
more functional groups such as glutaraldehyde, and toluene
diisocyanate; embedding in which the microorganism is embedded in a
polymer such as calcium alginate, carrageenan, and photo-setting
resins; and the like methods (see "Koteika Seitai Shokubai
(Immobilized Biocatalyst) pp. 67-81 (Kodansha Scientific K.K.).
In treatment of a liquid where diffusion of the microorganism into
the liquid medium is required to be prevented, a fixing method is
suitably selected which bonds the microorganism strongly to the
carrier. In this case, strong fixation will not cause a problem,
since mass transfer in liquid medium usually proceeds relatively
readily.
In treatment of soil, relatively loose fixation of microorganism to
the carrier is required so that the microorganism may readily come
off and migrate into the soil to come to contact with the polluting
substance effectively with the sufficient number of microorganism
being retained by the carrier. In this case, the carrier and
fixation state of the microorganism are selected so as to obtain a
relatively loose fixation state.
In the treatment of a polluted area of natural environment,
frequently the applied microorganism cannot function effectively
owing to competition with other microorganisms living already in
the natural ecosystem or predation of the applied microorganism by
protists or the like. Otherwise the applied microorganism cannot
frequently be predominant because of the oligotrophic conditions of
the environment to be treated.
Therefore against the severe external environment, the
microorganism may be protected by using a carrier having pores of
several .mu.m diameter and suitable depth and forming microhabitat
of the microorganism in the pore. For example, even if the outside
of the pore is in a dry state that adversely affects viability of
the microorganism, the microorganism can survive owing to the
capillary water retained in the microhabitat. Even when a salt
concentration suddenly changes outside the pores, the influence of
the change is weakened by the buffering effect, given by the
distance between the microhabitat and the external environment,
thereby protecting the microorganism in the microhabitat.
Further, invasion and extermination of the applied microorganism by
a native microorganism can be prevented by formation of a
predominating region of the applied microorganism in the
microhabitat. If the inside diameter of the pore of the
microhabitat is formed to have a diameter of less than about 10
.mu.m, the invasion and predation by protists can be prevented.
By formation of such microhabitat for the microorganism in the pore
of the carrier before application to the soil, the microorganism in
the pore can survive or grow protected from other native
microorganism in the soil or the soil environment. The
microorganism will be maintained for a longer period than the
microorganism directly applied to the soil. The microorganism is
released from the pore into the soil and it decomposes the
polluting substance in the soil.
The carrier may be in any shape provided that it has pores for
formation of microhabitat for the microorganism to be applied. The
diameter of the carrier is preferably in the range of from several
hundred .mu.m to several mm to facilitate the migration or
dispersion of the carrier in the soil.
The material for constructing the carrier includes inorganic
materials such as charcoal, porous ceramics, porous glass, calcium
silicate, silica, kaolinite, and montmorillonite; soil materials
having aggregate structure such as Kanuma soil; active carbon; and
organic materials such as urethane foams and anion-exchange
resins.
By use of a biodegradable material for formation of the carrier,
problems are eliminated regarding secondary pollution caused by
remaining carrier and damage of the ecosystem caused by the applied
microorganism. A biodegradable carrier is preferred which is
degraded gradually to remove the microhabitat after the remediation
treatment by the applied microorganism. By use of such a carrier,
for example, in soil treatment, the applied microorganism is
released into the soil as the result of disappearance of the
microhabitat, and decreases in number and finally becomes extinct
owing to competition with native microorganisms, predation by
protist, and severe conditions for growth. Thus, the ecosystem in
the soil being restored to the original state. Such effects may be
expected also in liquid treatment.
The material for such biodegradable carriers includes cellulose,
lignin, starch, agarose, dextran, albumin, chitin, chitosan, filter
paper, wood pieces, and so forth. The carrier made of such a
material is preferred since it fixes the microorganism relatively
loosely, releases the grown microorganism relatively readily, is
inexpensive, and in some cases, becomes a nutrient for the applied
microorganism itself.
The rate of degradation of the biodegradable carrier itself may be
controlled by selecting the kind and properties of the constituting
material. For example, the diameter and shape of the pores, the
size and shape of the carrier, and so forth are suitably selected
in consideration of the material. In selecting the above
requirements, the factors to be considered in connection with the
degradation rate include the kind, the amount and
carrier-degradation activity of the microorganism, and the volume
of the treated soil. The carrier is preferably designed after
confirming decomposition of the polluting substance, and
degradation of the carrier by field experiments.
The carrier contains preferably the required nutrient because the
contact of the applied microorganism with the required nutrient is
facilitated thereby and the control of the applied microorganism is
also readily conducted by control of the required nutrient.
Further, the working period of the applied microorganism and the
remaining period of the carrier can be controlled by employing at
least one required nutrient as the constituent of the carrier in a
controlled amount to cause degradation of the carrier by
consumption of the required nutrient by the applied microorganism.
In this case also, it is more desirable to make the entire carrier
biodegradable from the standpoint of environmental protection.
For treatment of a liquid, one example of a preferred type of
carrier has a required nutrient fixed thereon and has an applied
microorganism fixed such that the microorganism is not excessively
readily released into the liquid. This type of carrier which
contains the required nutrient facilitates the contact of the
microorganism with the nutrient and makes the supply of the
nutrient efficient. Furthermore, the microorganism does not diffuse
into the environment since both the required nutrient and the
applied microorganism are fixed on the carrier. With this type of
carrier, an auxotrophic microorganism diffusing into the
environment dies from the lack of the required nutrient. This is
suitable for the case where diffusion of the applied microorganism
causes a problem, and the release of the required nutrient to the
environment is suppressed.
In treatment of soil where mass transfer is slow, the treatment is
made more effective by the constitution which releases the applied
microorganism and the required nutrient fixed on the carrier into
the peripheral space. In an example, the carrier for forming a
microhabitat contains at least one required nutrient as a
constituent, and the carrier is gradually collapsed as the nutrient
is consumed by the applied microorganism, resulting finally in
collapse of the microhabitat as the result of degradation of the
carrier and leading to extinction of the applied microorganism.
This is suitable in the case where the remaining applied
microorganism in the soil causes a problem.
EXAMPLES
In Examples, M9 culture medium employed in the following examples
has the composition below.
M9 Culture Medium Composition (per liter):
______________________________________ NaH.sub.2 PO.sub.4 6.2 g
KH.sub.2 PO.sub.4 3.0 g NaCl 0.5 g NH.sub.4 Cl 1.0 g (pH 7.0)
______________________________________
The change of the number of bacteria was estimated from the change
of the light absorbance (O. D.)
Example 1
(Growth of Auxotrophic Microorganism with Required Nutrient,
Decomposition of Polluting Substance Thereby, and Fate of
Microorganism in Absence of Required Nutrient)
A leucine-requiring mutant was obtained by ultraviolet light
irradiation from Pseudomonas cepacia KK01 strain which is a novel
bacteria strain having an ability of decomposing a phenolic
compound such as phenol, o-cresol, m-cresol, and p-cresol, and was
deposited in Fermentation Research Institute of Agency of
Industrial Science and Technology of MITI of Japan on Mar. 11, 1992
having a depository address of 1-3, Higashi 1-chome, Tsukuba-shi,
Ibaraki-ken, 305, Japan, and converted to international deposition
on Mar. 9, 1993 under Budapest Agreement (Deposit No.: FERM
BP-4235). The isolated strain is the same as a KK01 strain
(International Deposition No. FERM BP-4235) that was deposited as a
novel strain having the power to degrade phenolic compounds such as
phenol, o-cresol, m-cresol and p-cresol in Fermentation Research
Institute, Agency of Industrial Science and Technology, Ministry of
International Trade and Industry on Mar. 11, 1992 and then changed
to the international deposition in accordance with the Budapest
treaty on Mar. 9, 1993.
A. Morphological Properties
(1) Gram stain: Negative
(2) Size and shape of the bacteria: Bacillus having a length of
1.0-2.0 .mu.m and a width of about 0.5 .mu.m.
(3) Mobility: Present
B. Growth state of the bacteria in each culture medium
______________________________________ Culture Growth Culture
Medium Temp. (.degree.C.) State
______________________________________ Blood agar culture medium 37
+ Lactose agar culture medium 37 + Chocolate agar culture medium 37
++ GMA 37 - Scyllo 37 -- Usual agar culture medium 4 - Usual agar
culture medium 25 .+-. Usual agar culture medium 37 - Usual agar
culture medium 41 .+-. ______________________________________
C. Physiological properties
(1) Aerobic or anaerobic: Strictly aerobic
(2) Degradation type of saccharose: Oxidation type
(3) Production of oxidase: +
(4) Reduction of silver nitrate: +
(5) Production of hydrogen sulfide: -
(6) Production of indole: -
(7) Production of urease: -
(8) Liquefaction of gelatin: -
(9) Hydrolysis of arginine: -
(10) Decarboxylation of lysine: +
(11) Decarboxylation of ornithine:
(12) Utilization of citric acid: +
(13) Methylcarbinolacetyl reaction (VP reaction): -
(14) Detection of tryptophane deaminase: -
(15) ONPG: -
(16) Utilization of carbohydrates:
Glucose: +
Fruit sugar: +
Maltose: +
Galactose: +
Xylose: +
Mannitol: .+-.
White sugar: -
Lactose: +
Aesculin: -
Inositol: -
Sorbitol: -
Rhamnose: -
Melibiose: -
Amygdalin: -
L-(+)-arabinose: +This mutant was inoculated on 5 ml of a culture
medium (M9 culture medium, additionally containing 0.05% of yeast
extract, 20 .mu.g/ml of leucine, and 500 ppm of phenol), and
incubated at 30.degree. C. When the culture came to exhibit the
value of O.D. of 0.7, the incubated culture was transferred to 500
ml of another culture medium (M9 culture medium containing 0.05% of
yeast extract and 500 ppm of phenol), and incubation was continued.
The required nutrient, leucine, was supplied in 10 ml (20 .mu.g/ml)
to the culture at an interval of 24 hours as shown in FIG. 1 by
arrow marks, and the daily changes of the number of bacteria and
the phenol concentration were estimated by light absorbance. After
48 hours, the liquid culture was divided into two equal parts, A
and B. The liquid culture A was incubated with supply of leucine in
the same manner as before, while the liquid culture B was incubated
without supply of leucine. The changes of number of the bacteria
and the phenol concentration were measured every 24 hours. FIG. 1
shows the results.
Example 2
(Bacterial Decomposition where the Required Nutrient is fixed in
Carrier)
The leucine-requiring mutant obtained from KK01 in Example 1 and
having phenol-decomposition ability was inoculated on 5 ml of a
culture medium (M9 culture medium, additionally containing 0.05% of
yeast extract, 20 .mu.g/ml of leucine, and 500 ppm of phenol), and
the culture was incubated at 30.degree. C. When the culture came to
exhibit the value of O.D. of 0.7, the liquid culture was further
incubated in a 500 ml scale until the O.D. value exceeded 0.7. The
liquid culture was centrifuged lightly, and the accumulated
bacterial mass was suspended in 20 ml of a culture medium (M9
additionally containing 20 .mu.g/ml of leucine). To the liquid
suspension, was added 24 ml of an aqueous solution of 7.5 g of
acrylamide monomer and 400 mg of N,N'-methylene-bis(acrylamide) as
a crosslinking agent, and the mixture was agitated. To the liquid
mixture, were added 1 ml of 25% .beta.-dimethylaminopropionitrile
as a polymerization accelerator and 5 ml of 1% K.sub.2 S.sub.2
O.sub.8 as a polymerization initiator at 8.degree. C. The mixture
was agitated well and kept at 20.degree. C. to obtain a
mutant-fixed carrier.
The obtained mutant-fixed carrier was crushed and dispersed in 500
ml of an aqueous 250-ppm phenol solution, and the dispersion was
incubated by standing at 25.degree. C. The change of the remaining
ratio of the phenol in the aqueous solution was determined by HPLC.
FIG. 2 shows the results.
Comparative Example 1
(Bacterial Decomposition without Required Nutrient Fixed on
Carrier)
The leucine-requiring mutant obtained from KK01 in Example 1 and
having phenol-decomposition ability was inoculated on 5 ml of a
culture medium (M9 culture medium, additionally containing 0.05% of
yeast extract, 20 .mu.g/ml of leucine, and 500 ppm of phenol), and
the culture was incubated at 30.degree. C. When the culture came to
exhibit the value of O.D. of 0.7, the liquid culture was further
incubated in a 500 ml scale until the O.D. value exceeded 0.7. The
liquid culture was centrifuged lightly, and the accumulated
bacterial mass was suspended in 20 ml of an M9 culture medium. To
the liquid suspension, was added 24 ml of an aqueous solution of
7.5 g of acrylamide monomer and 400 mg of
N,N'-methylene-bis(acrylamide) as a crosslinking agent, and mixed.
To the liquid mixture, were added 1 ml of 25%
.beta.-dimethylaminopropionitrile as a polymerization accelerator
and 5 ml of 1% K.sub.2 S.sub.2 O.sub.8 as a polymerization
initiator at 8.degree. C. The mixture was agitated well and kept at
20.degree. C. to obtain a mutant-fixed carrier.
The obtained mutant-fixed carrier was pulverized and dispersed in
500 ml of an aqueous 250-ppm phenol solution, and the dispersion
was incubated by standing at 25.degree. C. The change of the
remaining ratio of the phenol in the aqueous solution with lapse of
days was determined by HPLC. FIG. 2 shows the results.
Example 3
The leucine-requiring mutant obtained from KK01 in Example 1 and
having phenol-decomposition ability was inoculated on 5 ml of a
culture medium (M9 culture medium, additionally containing 0.05% of
yeast extract, 20 .mu.g/ml of leucine, and 500 ppm of phenol), and
the culture was incubated at 30.degree. C. When the culture came to
exhibit the value of O.D. of 0.7, the liquid culture was added to
500 ml of a culture medium (M9 culture medium, additionally
containing 0.05% of yeast extract, and 500 ppm of phenol) and was
further incubated until the O.D. value exceeded 0.7. In this
incubation, about 50 g in total of 3-mm cubic polyurethane pieces
were added and agitated for the purpose of promoting the growth of
the microorganism. The required nutrient, leucine, was supplied by
25 ml (20 .mu.g/ml) every 12 hours. The change of the remaining
ratio of the phenol in the aqueous solution with lapse of days was
determined by HPLC. FIG. 3 shows the results.
Comparative Example 2
The mutant was incubated in the same manner as in Example 3 except
that leucine was not supplied during the incubation in 500 ml
culture medium. The daily change of the phenol-remaining ratio was
measured. The results are shown in FIG. 3.
Example 4
(Growth of Auxotrophic Bacteria with Addition of Required Nutrient
)
The Leucine-requiring mutant of Pseudomonas cepacia KK01 from
Example 1 was inoculated on 5 ml of a liquid culture (M9 culture
medium, additionally containing 0.05% yeast extract and 20 .mu.g/ml
of leucine), and the culture was incubated at 20.degree. C. until
the O.D. of the culture exceeded 0.7. Then the liquid culture was
increased up to 500 ml, and the culture was further incubated until
the O.D. exceeded 0.7. The liquid culture was centrifuged lightly,
and the accumulated bacterial mass was dispersed in 500 g of
sterilized test soil. This test soil was incubated by standing at
25.degree. C. The required leucine was supplied by 50 ml (20
.mu.g/ml) every 24 hours.
The above experiment was conducted in five series: Run 1 to Run 5.
10 g of soil sample was taken from each test soil of the Runs. The
number of the bacteria of each soil was estimated by plate dilution
culture. The average of the estimated numbers of bacteria was
defined as the number of the bacteria. The change of the number of
the bacteria with lapse of time was estimated. FIG. 4 shows the
results.
Comparative Example 3
(Absence of Required Nutrient)
The change in the number of the bacteria was measured in the same
manner as in Example 4 except that leucine was not supplied to the
test soil. FIG. 4 shows the results.
Example 5
(Required Nutrient Being Incorporated in Carrier Capable of Forming
Microhabitat)
The Leucine-requiring mutant of Pseudomonas cepacia KK01 Example 1
was inoculated on 5 ml of a liquid culture (M9 culture medium,
additionally containing 0.05% yeast extract and 20 .mu.g/ml of
leucine), and the culture was incubated at 30.degree. C. until the
O.D. of the culture exceeded 0.7. Then the liquid culture was
increased up to 500 ml, and the culture was further incubated until
the O.D. exceeded 0.7. The liquid culture was centrifuged lightly,
and the accumulated bacterial mass was dispersed in 500 g of
sterilized test soil. Thereto filter paper which had been
impregnated with an aqueous leucine solution (80 .mu.g/ml) and
crushed into pieces of 3 mm or smaller was added in a total weight
of about 50 g, and the mixture was blended and incubated by
standing at 25.degree. C.
The daily change of the number of the bacteria in the soil was
estimated in the same manner as in Example 1. FIG. 5 shows the
results.
Comparative Example 4
(No Required Nutrient Being Incorporated in Carrier Capable of
Forming Microhabitat)
The incubation of the soil was conducted in the same manner as in
Example 5 except that the filter paper was not impregnated with the
aqueous leucine solution, and the daily change of the number of the
bacteria in the soil was estimated. FIG. 5 shows the results.
Example 6
(Bacterial Decomposition with Microhabitat Formed from Required
Nutrient)
The leucine-requiring mutant obtained in Example 1 was inoculated
on 5 ml of a culture medium (M9 culture medium, additionally
containing 0.05% of yeast extract, 20 .mu.g/ml of leucine, and 500
ppm of phenol), and the culture was incubated at 30.degree. C. When
the culture came to exhibit the value of O.D. of 0.7, the liquid
culture was further incubated in a 500 ml scale until the O.D.
value exceeded 0.7. The liquid culture was centrifuged lightly, and
the accumulated bacterial mass was dispersed in 500 g of sterilized
test soil in which 80 ml of an aqueous 250 ppm phenol solution had
been diffused. Thereto filter paper which had been impregnated with
an aqueous leucine solution (80 .mu.g/ml) and crushed into pieces
of 3 mm or smaller was added in a total weight of about 50 g, and
the mixture was blended and incubated by standing at 25.degree.
C.
The daily changes in the number of the bacteria and of the phenol
remaining ratio were estimated in the same manner as in Example 4.
The phenol in the soil was determined by HPLC. FIG. 6 shows the
results.
Comparative Example 5
(Bacterial Decomposition with Microhabitat Formed without Required
Nutrient)
The incubation of the soil was conducted in the same manner as in
Example 6 except that the filter paper was not impregnated with the
aqueous leucine solution, and the daily change of the number of the
bacteria in the soil was estimated. FIG. 7 shows the results.
Example 7
(Bacterial Decomposition within Polluted Area Only)
The leucine-requiring mutant obtained in Example 1 was inoculated
on 5 ml of a culture medium (M9 culture medium, additionally
containing 0.05% of yeast extract, 20 .mu.g/ml of leucine, and 500
ppm of phenol), and the culture was incubated at 30.degree. C. When
the culture came to exhibit the value of O.D. of 0.7, the liquid
culture was further incubated in a 500 ml scale until the O.D.
value exceeded 0.7. The liquid culture was centrifuged lightly, and
the accumulated bacterial mass was dispersed in 500 g of sterilized
test soil in which 80 ml of an aqueous 250-ppm phenol solution had
been diffused. Thereto filter paper which had been impregnated with
an aqueous leucine solution (80 .mu.g/ml) and crushed into pieces
of 3 mm or smaller was added in a total amount of about 50 g, and
the mixture was blended and incubated by standing at 25.degree. C.
for one day. The incubated soil having the microhabitat therein was
placed in non-microhabitat-containing sterilized test soil
(containing phenol in an amount of 0.5 ppm/g) as shown in FIG. 8,
and the entire sol was incubated by standing at 25.degree. C. In
FIG. 8, A is a sterilized test soil polluted and containing
microhabitat, and B is a sterilized test soil polluted and not
containing microhabitat. The change of the number of the bacteria
with lapse of time was estimated for both of the
microhabitat-containing region and the non-microhabitat-containing
region respectively. Similarly the phenol in the soil regions was
determined by HPLC to estimate the phenol remaining ratio. The
results for the microhabitat-containing soil are shown in FIG. 9,
and the results for the non-microhabitat-containing soil are shown
in FIG. 10.
Example 8
The leucine-requiring mutant obtained in Example 1 was inoculated
on 5 ml of a culture medium (M9 culture medium, additionally
containing 0.05% of yeast extract, 20 .mu.g/ml of leucine, and 500
ppm of phenol), and the culture was incubated at 30.degree. C. When
the culture came to exhibit the value of O.D. of 0.7, the liquid
culture was further incubated in a 500 ml scale until the O.D.
value exceeded 0.7. The liquid culture was centrifuged lightly, and
the accumulated bacterial mass was dispersed in 500 g of sterilized
test soil in which 80 ml of an aqueous 250 ppm phenol solution had
been diffused. The test soil was brown forest soil having been
picked at forest in Morinosato, Atsugi-Shi, Kanagawa-Ken, Japan.
Thereto 3-mm cubic polyurethane pieces were added in a total weight
of 50 g, and the mixture was blended and incubated by standing at
25.degree. C. The required leucine was supplied by 50 ml (20
.mu.g/ml) every 24 hours.
The phenol in the soil was determined by HPLC to estimate the
change of the remaining ratio. FIG. 11 shows the results.
Comparative Example 6
The remaining ratio of phenol was estimated in the same manner as
in Example 8 except that leucine was not supplied to the test soil.
FIG. 11 shows the results.
According to the present invention, microorganism works only in a
polluted area and the microorganism dies spontaneously after
remediation of pollution. Therefore, even when a microorganism is
used in an open system. For example in treatment of spillage oil by
microorganism on the sea, the present invention enables environment
protection without adverse effect caused by the remaining
microorganism after the treatment since the employed microorganism
dies after treatment.
* * * * *